Method and apparatus for reading reporter labeled beads

• US 6,934,408 B2
• Filed: 10/12/2001
• Issued: 08/23/2005
• Est. Priority Date: 08/25/2000
• Status: Expired due to Fees

• Current Assignee: Cytek Biosciences Inc
• Original Assignee: Amnis Corporation (DiaSorin S.p.A.)
• Inventors: Basiji, David A., Perry, David J., Ortyn, William E., Bauer, Richard A., Frost, Keith L., Finch, Rosalynde J.
• Primary Examiner(s): Mehta, Bhavesh M.
• Assistant Examiner(s): Choobin, Barry
• Application Number:
  US09/976,237
• Publication Number:
  US 20020094116A1
• Time in Patent Office: 1,411 Days
• Field of Search: 382/128, 382/129, 382/133, 382/134, 382/173, 435/6, 435/7.2, 356/317, 356/300, 356/39, 250/591, 128/922, 377/10, 600/310, 436/66
• US Class Current: 382/129
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 60/10 for identifying library members
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1447 Spatial selection
• CPC Class Codes: G01N 2015/1472 with colour
• CPC Class Codes: G01N 2015/1479 Using diffuse illumination or excitation
• CPC Class Codes: G01N 2015/1497 Particle shape
• CPC Class Codes: G02B 27/0012 Optical design, e.g. procedures, algorithms, optimisation routines
• CPC Class Codes: G02B 27/1013 for colour or multispectral image sensors, e.g. splitting an image into monochromatic image components on respective sensors spectral imaging systems G01J
• CPC Class Codes: G02B 27/126 The splitting element being a prism or prismatic array, including systems based on total internal reflection
• CPC Class Codes: G02B 27/144 using partially transparent surfaces without spectral selectivity G02B27/147 takes precedence
• CPC Class Codes: G02B 27/145 having sequential partially reflecting surfaces
• CPC Class Codes: G02B 27/148 including stacked surfaces having at least one double-pass partially reflecting surface
• CPC Class Codes: G02B 7/28 : Systems for automatic generation of focusing signals

Abstract

Combinatorially-synthesized deoxyribonucleic acid (DNA) oligonucleotides attached to encoded beads that are hybridized to amplified and labeled genomic DNA or ribonucleic acid (RNA) are analyzed using a flow imaging system. Oligonucleotides and corresponding reporters are bound to the surfaces of a plurality of small beads such that different beads bear different oligo sequences. Each bead bears a unique optical signature comprising a predefined number of unique reporters, where each reporter comprises a predefined combination of different fluorochromes. The composite spectral signature in turn identifies the unique nucleotide sequence of its attached oligo chains. This optical signature is rapidly decoded using an imaging system to discriminate the different reporters attached to each bead in a flow in regard to color and spatial position on the bead.

Claims

1. A method for imaging a plurality of encoded beads entrained in a flow of fluid to identify a compound attached to each encoded bead, corresponding reporters attached to each bead identifying a unique bead signature, thereby identifying the attached compound, said method comprising the steps of:

(a) providing an imaging system for imaging encoded beads contained within the flow of fluid, said imaging system including at least one light source for illuminating an encoded bead within the flow of fluid passing through the imaging system;

(b) focussing light from the encoded bead along a collection path that is in a direction not aligned with the flow of fluid;

(c) dispersing the light that is traveling along the collection path into a plurality of light beams, as a function of a plurality of different discriminable characteristics of the light, said plurality of different discriminable characteristics being indicative of an identity of each reporter that may be attached to the encoded bead;

(d) focussing each of the light beams to produce a respective image corresponding to that light beam;

(e) detecting respective images thus produced;

(f) generating a plurality of signals based on the respective images, each signal identifying those reporters present on each encoded bead;

(g) analyzing each respective image to determine the identity of each reporter present on each encoded bead, thereby identifying the compound attached to that bead; and

(h) repeating steps (a)-(g) for successive encoded beads in the flow of fluid.

2. The method of claim 1, wherein the step of analyzing each respective image comprises the steps of:

(a) determining a signature of each reporter associated with the encoded bead based upon the locations of the reporters on the bead; and

(b) identifying the compound as a function of each reporter associated with the encoded bead.

3. The method of claim 1, wherein the step of analyzing comprises the steps of disregarding images relating to a reporter if an image from an identical reporter have already been analyzed, and disregarding all images for an encoded bead if said images indicate that fewer than a predetermined number of reporters are associated with the encoded bead.

4. The method of claim 1, wherein the step of analyzing comprises the step of referring to an encoded bead legend that relates each unique set of reporters to a specific compound.

5. The method of claim 4, wherein the step of analyzing comprises the step of disregarding all images for an encoded bead if it is determined that the encoded bead does not correspond to said bead legend.

6. The method of claim 1, wherein the step of analyzing comprises the step of de-convolving the images if the step of dispersing convolves the plurality of light beams.

7. The method of claim 1, wherein the step of dispersing comprises the step of providing an image corresponding to a binding signal produced by the encoded bead itself.

8. The method of claim 7, further comprising the step of disregarding all signals for an encoded bead if any image relating to that encoded bead indicates that the encoded bead has not experienced a binding event.

9. A flow imaging system for sequentially imaging and decoding a plurality or encoded beads entrained in a fluid, said encoded beads comprising one or more compounds from among a plurality of different compounds, and one or more reporters from among a plurality of different reporters, each different compound being uniquely identified by at least one reporter, said flow imaging system comprising:

(a) at least one light source for illuminating an encoded bead within a flow of fluid passing through the flow imaging system;

(b) a collection lens disposed so that light traveling from an encoded bead passes through the collection lens and travels along a collection path;

(c) a dispersing component that receives the light from the collection lens and disperses the light into a plurality of light beams, as a function of a plurality of different discriminable characteristics of the light, said plurality of different discriminable characteristics being indicative of the plurality of different reporters;

(d) at least one pixilated detector;

(e) an imaging lens that focuses each of the plurality of light beams on said at least one pixilated detector, producing a respective image corresponding to each of the plurality of light beams, said at least one pixilated detector providing an output signal for each respective image, each output signal identifying those reporters present on the encoded bead; and

(f) a signal processor coupled to receive the output signals from said at least one pixilated detector, said signal processor processing the output signals to decode those compounds present on the encoded bead, based on the identity of those reporters present on the encoded bead.

10. The flow imaging system of claim 9, wherein said at least one pixilated detector comprises a time delay and integration (TDI) detector.

11. The flow imaging system of claim 9, wherein said signal processor is adapted to analyze said output signals to:

(a) determine a signature of each reporter associated with the encoded bead based upon a locations of the reporters on the bead; and

(b) identify the compounds as a function of each reporter associated with the encoded bead.

12. The flow imaging system of claim 9, wherein said signal processor is adapted to disregard all output signals relating to a reporter if signals from an identical reporter have already been analyzed, and to disregard all output signals for an encoded bead if said signals indicate that fewer than a predetermined number of reporters are associated with the encoded bead.

13. The flow imaging system of claim 9, wherein said signal processor is adapted to de-convolving the output signals if said dispersing component convolves the plurality of light beams.

14. The flow imaging system of claim 9, wherein said dispersing component provides one respective image corresponding to a binding signal produced by the encoded bead itself.

15. The flow imaging system of claim 14, wherein said signal processor is adapted to disregard all output signals for an encoded bead if said one respective image indicates that an encoded bead has not experienced a binding event.

16. A flow imaging system for sequentially imaging and decoding a plurality of encoded beads entrained in a fluid, said encoded beads comprising one or more compounds from among a plurality of different compounds, and one or more reporters from among a plurality of different reporters, each compound being uniquely identified by at least one reporter, said flow imaging system comprising:

(a) a collection lens disposed so that light traveling from an encoded bead passes through the collection lens and travels along a collection path;

(b) a plurality of light reflecting elements disposed in the collection path, each light reflecting element reflecting light of a different predefined characteristic, and passing light that does not have that predefined characteristic, the reporters on an encoded bead determining the characteristics of light traveling along the collection path, each light reflecting element being positioned at a different location with respect to the collection path to reflect light of a specific predefined characteristic in a direction different from that of other light reflecting elements, each light reflecting element being positioned along an axis of said collection path, such that passing light not reflected by a preceding light reflecting element reaches a last light reflecting element;

(c) at least one pixilated detector disposed to receive light that has been reflected by each of the light reflecting elements, said at least one pixilated detector comprising a plurality of pixilated regions, each pixilated region producing an output signal that is indicative of at least one characteristic of the encoded beads and thus indicative of the reporters; and

(d) a signal processor coupled to receive the output signals from said the plurality of regions, said signal processor processing the output signals to decode an identity of the compounds as a function of the reporters present on the encoded bead.

17. The flow imaging system of claim 16, wherein said plurality of light reflecting elements comprise dichroic filters.

18. A flow imaging system for sequentially imaging and decoding a plurality of encoded beads entrained in a fluid, said encoded beads comprising one or more compounds from among a plurality of different compounds, and one or more reporters from among a plurality of different reporters, each compound being uniquely identified by at least one reporter, said flow imaging system comprising:

(a) a collection lens disposed so that light traveling from an encoded bead object passes through the collection lens and travels along a collection path;

(b) a dispersing component that receives the light from the collection lens and disperses the light into a plurality of light beams, as a function of a plurality of different discriminable characteristics of the light, said plurality of different discriminable characteristics being indicative of the plurality of different reporters;

(c) a plurality of light sensitive regions disposed on at least one detector;

(d) an imaging lens that focuses each of the plurality of light beams on said plurality of light sensitive regions, producing a respective image corresponding to each of the plurality of light beams, said plurality of light sensitive regions providing an output signal for each respective image, each output signal indicating those reporters present on the encoded bead; and

(e) means for processing the output signals to decode a sequence of the plurality of components.

19. The flow imaging system of claim 18, wherein said means comprise a signal processor coupled to receive the output signals from said at plurality of regions.

20. The flow imaging system of claim 18, wherein said means decodes a sequence of the plurality of components by:

(a) determining dispositions of the reporters on the encoded bead;

(b) determining a signature of each reporter associated with the encoded bead based upon the dispositions of the reporters on the bead; and

(c) identifying each compound as a function of each reporter associated with the encoded bead.

21. The flow imaging system of claim 18, wherein said means disregards all output signals relating to a reporter if signals from an identical reporter have already been analyzed for the encoded bead, and disregards all output signals for an encoded bead if said signals indicate that fewer than a predetermined number of reporters are associated with the encoded bead.

22. The flow imaging system of claim 18, wherein said means de-convolves the output signals if said dispersing component convolves the plurality of light beams.

23. A flow imaging system for sequentially imaging and decoding a plurality of encoded beads entrained in a fluid, said encoded beads being associated with one or more compounds from among a plurality of different compounds, and with one or more reporters from among a plurality of different reporters, each compound being uniquely identified by at least one reporter, said flow imaging system comprising:

(a) a fluid system comprising an unanalyzed encoded bead supply, a detection volume, and an analyzed encoded bead reservoir, said fluid system being specifically adapted to hydrodynamically focus fluid moving from said unanalyzed encoded bead supply into said detection volume, such that encoded beads pass through said detection volume one at a time and into the encoded bead reservoir;

(b) means for collecting image data from each encoded bead passing though said detection volume, said image data being indicative of at least one characteristic of the encoded bead passing through the detection volume that is determinative of the reporters associated with the encoded bead; and

(c) a signal processor capable of decoding a plurality of encoded beads based on said at least one characteristic of the encoded beads collected by said means, to determine the compounds associated with each encoded bead that has been analyzed.

24. The flow imaging system of claim 23, wherein said means for collecting image data from each encoded bead comprises:

(a) a collection lens disposed so that light traveling from the encoded beads passes through the collection lens and travels along a collection path;

(b) a dispersing component that receives the light from the collection lens and disperses the light into a plurality of light beams, as a function of a plurality different discriminable characteristics of the light, said plurality of different discriminable characteristics being indicative of the plurality of different reporters;

(c) at least one pixilated detector;

(d) an imaging lens that focuses each of the plurality of light beams on said at least one pixilated detector, producing a respective image corresponding to each of the plurality of light beams, said at least one pixilated detector providing an output signal for each respective image, each output signal indicating those reporters present on the encoded bead; and

(e) a signal processor coupled to receive the output signals from said at least plurality of pixilated detectors, said signal processor processing the output signals to decode an identity of the compounds associated with each encoded bead analyzed.

25. The flow imaging system of claim 23, wherein said signal processor is adapted to analyze said output signals to:

(a) determine locations of the reporters on the encoded bead,

(b) determine a signature of each reporter associated with the encoded bead based upon the locations of the reporters on the bead; and

(c) identify the compounds associated with each encoded bead as a function of each reporter associated with each encoded bead analyzed.

26. The flow imaging system of claim 23, wherein said signal processor is adapted to disregard all output signals relating to a reporter if signals from an identical reporter have already been analyzed, and to disregard all output signals for an encoded bead if said signals indicate that fewer than a predetermined number of reporters are associated with the encoded bead.

27. The flow imaging system of claim 23, wherein said signal processor is adapted to de-convolving the output signals if said dispersing component convolves the plurality of light beams.

28. The flow imaging system of claim 23, wherein said dispersing component provides one respective image corresponding to a binding signal produced by the encoded bead itself.

29. The flow imaging system of claim 28, wherein said signal processor is adapted to disregard all output signals for an encoded bead if said one respective image indicates that an encoded bead has not experienced a binding event.

30. A method of employing an oligo library encoded on beads for at least one of a DNA sequencing, a polymorphism analysis, and an expression analysis, said method comprising the steps of:

(a) providing an imaging system capable of decoding a sequence of encoded beads conveyed in a flow of fluid;

(b) generating a complete encoded bead library of N-mer oligos;

(c) selectively performing one of said DNA sequencing, said polymorphism analysis, and said expression analysis based on imaging data produced by imaging the encoded beads with the imaging system;

(d) when the DNA sequencing is selected, amplifying genomic DNA using primers for extended sequences of interest;

(e) when the polymorphism analysis is selected, amplifying genomic DNA using primers for polymorphic regions of interest;

(f) when the expression analysis is selected, amplifying RNA using primers for genes of interest;

(g) hybridizing an amplified component produced by any of steps (d)-(f) in relationship to said encoded bead library;

(h) analyzing the encoded beads using the imaging data to identify oligo sequences of encoded beads hybridized in step (g); and

(i) constructing sequence contigs from the oligo sequences identified in step (h) to identify one of a genomic DNA sequence, a polymorphic allele, and an expressed gene.

31. The method of claim 30, wherein said N-mer oligos comprises oligos having a length equal to ten.